It is considered that sugar chains are related to vital phenomena such as development, differentiation and cell recognition and also deeply related to the development and progress of inflammations, cancers, autoimmune diseases and a large number of other diseases [Fukuda, M., Cell Surface Carbohydrates and Cell Development, CRC Press, Bosa Raton, Fla. (1992), Glycobiology, 3, 97 (1993)].
Sugar chains are present not only as glycoproteins, proteoglycans or glycolipids by addition to proteins or lipids and but also as oligosaccharides.
Gal β1,3-N-acetylglucosaminyltransferase is an enzyme having an activity of transferring N-acetylglucosamine to galactose residues present at the non-reducing terminal of a sugar chain via a β1,3-linkage, and involved in the synthesis of a sugar chain having a GlcNAc β1-3Gal structure. Sugar chains having a GlcNAcβ1-3Gal structure are present in N-glycosylated sugar chains and O-glycosylated sugar chains of glycoproteins and also in neolacto-series and facto-series glycolipids, and in oligosaccharides.
With regard to a Gal β1,3-N-acetylglucosaminyltransferase, its partial purification has so far been reported [J. Biol. Chem., 268, 27118 (1993), J. Biol. Chem., 267, 2994 (1992), J. Biol. Chem., 263, 12461 (1988), Jpn. J. Med. Sci. Biol., 42, 77 (1989)]. Also, two types of Gal β1,3-N-acetylglucosaminyltransferase have been cloned [Proc. Natl. Acad. Sci. USA., 94, 14294 (1997), Proc. Natl. Acad. Sci. USA., 96, 406 (1999)]. The presence of other types of Gal β1,3-N-acetylglucosaminyltransferases has not been clarified.
Since sugar chains having a GlcNAcβ1-3Gal structure are present in great numbers, it is considered that two or more enzymes having different receptor substrate specificity or expressing at different tissue are present as Gal β1,3-N-acetylglucosaminyltransferases, and they are respectively taking part in different functions. Thus, it is an important subject to clone a Gal β1,3-N-acetylglucosaminyltransferase different from the two enzymes so far cloned, to examine its receptor substrate specificity as well as expression and distribution, and to elucidate its relation to biological functions and diseases.
It is known that lacto-N-neotetraose (Galβ1-4GlcNAβ1-3Galβ1-4Glc) and lacto-N-tetraose (Galβ1-3GlcNAβ1-3Galβ1-4Glc) or various oligosaccharides containing them as the core structure are present in human milk [Acta Paediatrica, 82, 903 (1993)]. It is considered that these oligosaccharides have a function of preventing babies from viral and microbial infections and a function of neutralizing toxins. Also, they have an activity of accelerating growth of Bifidobacterium that is a good enteric bacterium.
It will be markedly useful from the industrial point of view if these oligosaccharides contained in human milk or milk containing them can be efficiently produced. If a gene for a Gal β1,3-N-acetylglucosaminyltransferase involved in the synthesis of these oligosaccharides contained in human milk is obtained, these oligosaccharides would be efficiently synthesized, but such an enzyme has not been found.
Among a large number of sugar chains having a GlcNAcβ1-3Gal structure, particularly poly-N-acetyllactosamine sugar chains serve as core sugar chains of many functional sugar chains (selectin ligand sugar chains, microbial or viral receptor sugar chains, SSEA-1 sugar chains, cancer-related sugar chains and the like) and deeply relate to diseases, such as embryogenesis, cell differentiation or diseases such as inflammation, cancer and the like.
Since there is a possibility that Gal β1,3-N-acetylglucosaminyltransferases involved in the synthesis of poly-N-acetyllactosamine sugar chains functioning in respective cases are different, it is an important subject to clone a Gal β1,3-N-acetylglucosaminyltransferase which is different from the two enzymes so far cloned and to estimate functions of respective enzymes based on their receptor substrate specificity, expression distribution and the like.
The poly-N-acetyllactosamine sugar chain is synthesized by the alternate functions of a GlcNAc β1,4-N-galactosyltransferase activity and a Gal β1,3-N-acetylglucosaminyltransferase activity. Regarding β1,4-galactosyltransferases, genes for 4 enzymes (β4Gal-T1, β4Gal-T2, β4Gal-T3 and β4Gal-T4) have been cloned, and the receptor substrate specificity of each enzyme has been analyzed [J. Biol. Chem., 272, 31979 (1997), J. Biol. Chem., 273, 29331 (1997)].
Accordingly, a poly-N-acetyllactosamine sugar chain can be synthesized in vitro by using a GlcNAc β1,4-galactosyltransferase and a Gal β1,3-N-acetylglucosaminyltransferase. Also, a poly-N-acetyllactosamine sugar chain or a complex carbohydrate to which the sugar chain is added can be synthesized by co-expressing a GlcNAc β1,4-galactosyltransferase gene and a Gal β1,3-N-acetylglucosaminyltransferase gene in cells.
Since a GlcNAc β1,4-galactosyltransferase is expressed in almost all cells, poly-N-acetyllactosamine sugar chain or a sugar to which the sugar chain is added can be synthesized by expressing a Gal β1,3-N-acetylglucosaminyltransferase gene in cells.
It is known that a poly-N-acetyllactosamine sugar chain is more frequently expressed in cancer cells in comparison with corresponding normal cells [J. Biol. Chem., 259, 10834 (1984), J. Biol. Chem., 261, 10772 (1986), J. Biol. Chem., 266, 1772 (1991), J. Biol. Chem., 267, 5700 (1992)].
It is expected that a poly-N-acetyllactosamine sugar chain having sialyl Lewis x sugar chain might be a medicament having an anti-inflammatory effect or a metastasis inhibiting effect, as a selectin antagonist.
A partially purified β1,3-N-acetylglucosaminyltransferase has been used in synthesizing the poly-N-acetyllactosamine sugar chain moiety of these oligosaccharides. But since supply of this enzyme is a rate-limiting step, it is difficult to synthesize the poly-N-acetyllactosamine sugar chain in a large amount [Glycobiology, 7, 453 (1997)].
On the other hand, a poly-N-acetyllactosamine sugar chain can also be synthesized by chemical synthesis, but its synthesis requires considerably complex steps [Tetrahedron Letter, 24, 5223 (1997)].
Thus, an efficient process for synthesizing a poly-N-acetyllactosamine sugar chain is in demand. Although two types of Gal β1,3-N-acetylglucosaminyltransferase so far cloned and genes for these enzymes can be used, it is considered that the use of other Gal β1,3-N-acetylglucosaminyltransferases having different substrate specificity and different function or their genes may be efficient in some cases depending on the purpose.
Since a poly-N-acetyllactosamine sugar chain contributes to the stabilization of protein [J. Biol. Chem., 265, 20476 (1990)], it is considered that a protein can be stabilized by artificially adding a poly-N-acetyllactosamine sugar chain to a desired protein. Also, since a clearance rate of proteins in blood from the kidney becomes slower as the effective molecular weight of the protein becomes larger, it is considered that the clearance rate from the kidney can be reduced and blood stability can be increased by artificially adding a poly-N-acetyllactosamine sugar chain to a desired protein to thereby increase the size of the effective molecular weight. In addition, there is a possibility that a desired protein can be targeted to a specified cell. Cases in which synthesizing ability of a poly-N-acetyllactosamine sugar chain has been increased are shown below.
It is shown that a poly-N-acetyllactosamine sugar chain is added to sugar chains of a membrane-bound glycoprotein of cells when F9 cell is treated with retinoic acid or Swiss 3T3 cell is treated with TGF-β [J. Biol. Chem., 268, 1242 (1993), Biochim. Biophys. Acta., 1221, 330 (1994)].
It is shown that, when N-ras proto-oncogene is expressed in NIH3T3 cell, activities of β1,4-galactosyltransferase and β1,3-N-acetylglucosaminyltransferase which are involved in the synthesis of poly-N-acetyllactosamine sugar chains are increased and the amount of poly-N-acetyllactosamine sugar chains in the N-linked sugar chain of a membrane protein is increased [J. Biol. Chem., 266, 21674 (1991)].
When the gene for core 2 β1,6-N-acetylglucosaminyltransferase is expressed in T-cell line EL-4, the molecular weight of a cell surface membrane protein CD43, CD45 or CD44 is increased [J. Biol. Chem., 271, 18732 (1996)]. The phenomenon is considered to be due to that the sugar chain synthesized by the core 2 β1,6-N-acetylglucosaminyltransferase becomes a good substrate of a β1,3-N-acetylglucosaminyltransferase which is involved in the synthesis of a oly-N-acetyllactosamine sugar chain.
Also, it is known that when HL-60 cell is cultured at 27° C., the amount of poly-N-acetyllactosamine sugar chains added to lamp-1 or lamp-2 is increased [J. Biol. Chem., 266, 23185 (1991)].
However, there are no reports on the efficient production of recombinant glycoproteins to which a poly-N-acetyllactosamine sugar chain is added, in host cells suitable for the production of recombinant glycoproteins (e.g., Namalwa cell, Namalwa KJM-1 cell, CHO cell and the like). Accordingly, development of a process for producing a recombinant glycoprotein to which a poly-N-acetyllactosamine sugar chain is added is an industrially important subject.
When mechanisms of inflammatory reaction and metastasis are taken into consideration, it is expected that inflammatory reaction can be inhibited and metastasis can be prevented by inhibiting expression of poly-N-acetyllactosamine sugar chain on leukocytes and cancer cells. If a gene for a Gal β1,3-N-acetylglucosaminyltransferase which is involved in the synthesis of a poly-N-acetyllactosamine sugar chain on leukocytes and cancer cells can be obtained, there is a possibility that expression of the poly-N-acetyllactosamine sugar chain on leukocytes and cancer cells can be inhibited by inhibiting expression of the gene.
Also, if a gene for a Gal β1,3-N-acetylglucosaminyltransferase which is involved in the synthesis of a poly-N-acetyllactosamine sugar chain on leukocytes and cancer cells can be obtained, there is a possibility that inflammatory diseases and the malignancy of cancers can be diagnosed by examining the expression level of the gene or examining the expression level of a protein encoded by the gene.
Since it is considered that Gal β1,3-N-acetylglucosaminyltransferases involved in the synthesis of a poly-N-acetyllactosamine sugar chain on specified leukocytes and cancer cells are different, it is necessary to clone and examine an enzyme different from the enzymes so far cloned.
Each of Gal β1,3-N-acetylglucosaminyltransferases expressed in a cell or tissue in which two or more Gal β1,3-N-acetylglucosaminyltransferases are expressed cannot be specified, and enzymological characteristics of each of the Gal β1,3-N-acetylglucosaminyltransferases cannot be clarified by enzymological analyses in which extracts of cells or tissues are used as enzyme sources.
In order to detect the expression of a specified Gal β1,3-N-acetylglucosaminyltransferase, it is necessary to use an immunological detection method by using a specific antibody or a detection method based on the nucleotide sequence of the gene (e.g., Northern hybridization or PCR). Accordingly, it is necessary to clone a Gal β1,3-N-acetylglucosaminyltransferase different from the enzymes so far cloned and compare their expressions.